Thiophene-2-carboximidamide Based Selective Neuronal Nitric Oxide Inhibitors

ABSTRACT

Selective neuronal nitric oxide synthase (nNOS) inhibitor compounds designed with one or more thiophene-2-carboximidamide substituents for improved bioavailability.

This application is a continuation of and claims priority benefit ofapplication Ser. No. 14/015, 551 filed Aug. 30, 2013 and issued as U.S.Pat. No. 8,735,606 on May 27, 2014, which claimed priority benefit fromapplication Ser. No. 61/695,187 filed Aug. 30, 2012, application Ser.No. 61/698,249 filed Sep. 7, 2012 and application Ser. No. 61/774,926filed Mar. 8, 2013—each of which is incorporated herein by reference inits entirety.

This invention was made with government support under grant numberGM049725 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND OF THE INVENTION

Neuronal nitric oxide synthase (nNOS) catalyzes the oxidation ofL-arginine to L-citrulline in the central nervous system, generatingnitric oxide (NO), a critical neurotransmitter. Significant research hasimplicated the overexpression of nNOS—and overproduction of NO—invarious neurological diseases, including Parkinson's, Alzheimer's, andHuntington's diseases, as well as neuronal damage due to stroke,cerebral palsy and migraine headaches. Inhibiting endothelial nitricoxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) is,however, undesirable, because these isozymes are responsible formaintaining crucial body function. Thus, selective inhibition of nNOSover its closely related isoforms, eNOS and iNOS, has provided apromising strategy in the development of therapeutics for the treatmentof neurodegenerative diseases. However, while certain compounds haveexhibited good potency and high selectivity, they often suffer from poorbioavailability. As a result, there remains an on-going search in theart for effective bioavailable NOS inhibitors to realize the therapeuticpotential of such compounds.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention toprovide compounds, compositions and related methods of use for theselective inhibition of neuronal nitric oxide synthase, therebyovercoming various deficiencies and shortcomings of the prior artincluding those outlined above. It would be understood by those skilledin the art that one or more aspects of this invention can meet certainobjectives, while one or more other aspects can meet certain otherobjectives. Each objective may not apply equally, in all its respects,to every aspect of this invention. As such, the following objects can beviewed in the alternative with respect to any one aspect of thisinvention.

It is an object of the present invention to provide one or more smallmolecule non-peptide compounds exhibiting selective nNOS inhibition overother enzyme isoforms and providing improved bioavailability—includingcompounds that are orally active.

It can be another object of the present invention to provide one or moresuch compounds for in vitro use and study under conditions promotingnitric oxide production, indicative of one or more mammalian diseasestates.

Alternatively, it can also be an object of the present invention toprovide one or more such compounds enabling in vivo treatment of suchdisease states.

Other objects, features, benefits and advantages of the presentinvention will be apparent from this summary and the followingdescriptions of certain embodiments of such compounds, compositionsand/or methods and will be readily apparent to those skilled in the arthaving knowledge of the synthetic techniques described herein. Suchobjectives, features, benefits and advantages will be apparent from theabove as taken into conjunction with the accompanying examples, data,figures and references incorporated herein, together with all reasonableinferences to be drawn therefrom.

In part, the present invention can be directed to compounds of a formula

wherein L can be a covalent bond or selected from divalent moieties suchas but not limited to CH═CH, CH₂CH₂, OCH₂CH(OH), O(CH₂)₂O, O(CH₂)₃O,(CH₂)₂NH, (CH₂)₂NHCH₂, CH₂CH(CH₂NH₂)CH₂CH₂, CH₂CH(CH₂NH₂)NHCH₂ andOCH(CH₂NH₂)CH₂O, together with salts, hydrates and/or solvates of suchcompounds.

Regardless of the identity of L, each phenyl moiety of such a compoundcan, independently, be substituted with a thiophene-2-carboximidamidemoiety either meta or para with respect to such a linker moiety. Incertain embodiments, such substituents can have a meta-relationship tosuch a linker and compounds can be of a formula

wherein L can be selected from divalent linker moieties of the sortdescribed above or referenced elsewhere herein.

In part, the present invention can also be directed to compounds of aformula

wherein n can be an integer selected from 1 and 2; R can be selectedfrom but not limited to

moieties; and R′ can be selected from but not limited to

moieties, together with salts, hydrates and/or solvates of suchcompounds.

In certain embodiments, the phenyl moiety of such a compound can bemeta-substituted. Regardless, such a compound can be of a formula

In part, the present invention can be directed to a compound selectedfrom compounds of a formula

wherein n can be an integer selected from 1 and 2, R can be

where R′ can be selected from H, alkyl, arylalkyl, alkenyl, arylalkenyl,alkynyl and arylalkynyl moieties, and X can be selected from H, halogen,methyl, mono- and polyfluoro-substituted methyl moieties; and compoundsof a formula

wherein R′ can be selected from H, methylene, alkyl, arylalkyl, alkenyl,arylalkenyl, alkynyl, and arylalkynyl moieties, and R″ can be selectedfrom H, a valence bond and a methylene moiety, providing that where R″is a valence bond or a methylene moiety, R′ can be a methylene moietywhereby R′ and R″ taken together can form a 5-6 member heterocyclicring, and X can be selected from H, halogen, methyl, mono- andpolyfluorosubstituted methyl moieties; and salts thereof. While X isshown, above, to denote monosubstitution, the phenyl moiety can also bepoly-substituted (e.g., X₁, X₂ and X₃, etc.) with one or a combinationof such substituents.

In certain non-limiting embodiments, R″ can be H and such a compound canbe of a formula

wherein R′ and X can be as discussed above. Alternatively, in certainother embodiments, at least one of R′ and R″ can be methylene, m can be0 or 1, and such a compound can be of a formula

In part, the present invention can also be directed to compoundsselected from compounds of a formula

and compounds of a formula

wherein, for each, L can be selected from divalent moieties of the sortdiscussed above or described elsewhere herein, together with salt,hydrates and/or solvates of such compounds. Without limitation, incertain such embodiments, for compounds of formula IV, L can be CH₂CH₂;and for compounds of formula V, L can be CH(OH)CH(OH).

In part, the present invention can also be directed to compounds of aformula

wherein n and n′ can, independently, be an integer selected from 0 and1, together with salts, hydrates and/or solvates of such compounds.

In part, the present invention can also be directed to compoundsselected from compounds of a formula

together with salts, hydrates and/or solvates of such compounds.

More generally, the present invention can also be directed to compoundsselected from compounds of a formula

and compounds of a formulawherein, for each, n and n′ can, independently, be an integer selectedfrom 0 and 1; for each, X can be selected from O and NH; and, for each,R can be selected from but not limited to phenyl, fluoro-, chloro- andbromo- and thiophene-2-carboximidamide substituted phenyl moieties,whether having an ortho-, meta-, or para-relationship to the prolinyllinker moiety, together with salts, hydrates and/or solvates of suchcompounds.

In certain embodiments, X can be 0. In certain such embodiments, n andn′ can be 0. Regardless, R can be a thiophene-2-carboximidamidesubstituted phenyl moiety. Without limitation, such a compound can bepresent as a trans stereoisomer. In certain other such embodiments, nand n′ can be 1. Regardless, R can be athiophene-2-carboximidamide-substituted phenyl moiety. Such a compoundcan be selected from the cis and trans stereoisomers.

The present compounds and, in particular the compounds of formulaeVI-VIII, are without stereochemical limitation. Where such compoundsand/or their intermediates are available as racemic mixtures, therespective isomers can be resolved. Likewise, as certain such compoundsare diastereomers, the corresponding enantiomers can be separated.Accordingly, any such stereocenter can be (S) or (R) with respect to anyother stereocenter(s). Regardless of stereochemistry, in certainembodiments, a compound of this invention can comprise a primary,secondary and/or tertiary amine and can be present as an acid salt,either partially or fully protonated. In certain such embodiments, acounter ion(s) can be a conjugate base of a protic acid. Whether or nota salt, one or more such compounds can be utilized as a component of apharmaceutical composition, optionally together with apharmaceutically-acceptable carrier component.

In part, the present invention can also provide a method of modulatingor affecting activity of inhibiting a nitric oxide synthase, such amethod comprising contacting a nitric oxide synthase with an effectiveamount of any one or more of the present compounds, including, but notlimited to those illustrated by the following examples, referencedfigures and/or accompanying synthetic schemes. Such a method cancomprise providing a compound or a related composition of thisinvention; and contacting a nitric oxide synthase enzyme with such acompound/composition, such contact as can selectively inhibit neuronalnitric oxide synthase over inducible and/or endothelial isoforms.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Chemical structures of representative, non-limiting inhibitorcompounds, in accordance with certain embodiments of this invention.

FIGS. 2A-B. Representative schematic chemical structures of non-limitingselective nNOS inhibitors compounds, in accordance with certainembodiments of this invention.

FIG. 3. Chemical structures of representative, non-limiting inhibitorcompounds, in accordance with certain embodiments of this invention.

FIG. 4. Representative schematic chemical structures of non-limitingselective nNOS inhibitor compounds in accordance with certainembodiments of this invention.

FIG. 5. Chemical structures of non-limiting inhibitor compounds andcorresponding synthetic intermediates, in accordance with certainembodiments of this invention.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Without limitation, in accordance with broader aspects of thisinvention, various compounds of this invention can be prepared asschematically illustrated, below. In particular, selective nNOSinhibitor compounds of this invention can be prepared using synthetictechniques common to the chemical literature and well-known to thoseskilled in the art. For instance, without limitation, a synthetic routefor target molecule 1 (FIG. 1) is shown in Scheme 1. Homo-couplingreaction of 1-iodo-3-nitrobenzene gave 26 in a moderate yield. The nitrogroups of 26 were reduced to amino groups in a quantitative yield, thena coupling reaction with methyl thiophene-2-carbimidothioate hydroiodidesalt was used to generate final product 1.

A synthetic route for target molecules 2 and 3 (FIG. 1) is shown inScheme 2. A Wittig reaction of 3-nitrobenzaldehyde with correspondingphosphorus glide of 1-(bromomethyl)-3-nitrobenzene allowed the isolationof the intermediate alkene in an 87% yield. The nitro groups of 29 werereduced to amino groups in a quantitative yield, and then a couplingreaction with methyl thiophene-2-carbimidothioate hydroiodide salt wasused to generate final product 2. Catalytic hydrogenation of 2 reducedthe double bond, giving final product 3 in a high yield.

A synthetic route for target molecule 4 (FIG. 1) is shown in Scheme 3. Anucleophilic addition reaction of 3-nitrophenol with2-bromo-1-(3-nitrophenyl)ethanone generated 31. The carbonyl group of 31was reduced to a hydroxyl group. The nitro groups of 32 were reduced toamino groups in a quantitative yield, and then a coupling reaction withmethyl thiophene-2-carbimidothioate hydroiodide salt was used togenerate final product 4.

A synthetic route for target molecules 5 and 6 (FIG. 1) is shown inScheme 4. Nucleophilic addition reactions of 3-nitrophenol withcorresponding alkyl halides generated 34 and 35. The nitro groups of 34and 35 were reduced to amino groups in quantitative yields, and thencoupling reactions with methyl thiophene-2-carbimidothioate hydroiodidesalt were used to generate final products 5 and 6, respectively.

A synthetic route for target molecules 7 and 8 (FIG. 1) is shown inScheme 5. Reductive amination of 3-nitrobenzaldehyde with correspondingamines generated 38 and 39. The amino groups of 38 and 39 were protectedby Boc group. The nitro groups of 40 and 41 were reduced to amino groupsin quantitative yields, and then coupling reactions with methylthiophene-2-carbimidothioate hydroiodide salt were used to generateintermediates 44 and 45, respectively. Deprotection of the Boc groupsgenerated 7 and 8.

A synthetic route for target molecule 9 (FIG. 1) is shown in Scheme 6.tert-Butyl 3-hydroxypyrrolidine-1-carboxylate was treated with NaH, andresulting anion was allowed to react with1-(bromomethyl)-3-nitrobenzene, giving ether 46 in a yield of 76%. Thenitro group of 46 was reduced to amino group in quantitative yield, andthen coupling reaction with methyl thiophene-2-carbimidothioatehydroiodide salt was used to generate intermediate 48. Deprotection ofthe Boc group generated final product 9. Analogously, as illustrated inScheme 6, various other nitrobenzene intermediates can be prepared enroute to the corresponding thiophene-2-carboximidamide. (See, e.g., FIG.5.)

A synthetic route for target molecule 10 (FIG. 1) is shown in Scheme 7.The hydroxyl group of tert-Butyl 3-hydroxypyrrolidine-1-carboxylate wasoxidized to aldehyde 49 with IBX, and then a reductive amination withcorresponding amine was used to generate 50. The amino group of 50 wasprotected by Boc group. The nitro group of 51 was reduced to amino groupin quantitative yield, and then coupling reaction with methylthiophene-2-carbimidothioate hydroiodide salt was used to generateintermediate 52. Deprotection of the Boc groups generated final product10.

A synthetic route for target molecules 11 and 12 (FIG. 1) is shown inScheme 8. Selected amines were subjected to amidation with dansylchloride to afford corresponding intermediates 54 and 55. The nitrogroups of 56 and 57 were reduced to amino groups in quantitative yields,and then coupling reactions with methyl thiophene-2-carbimidothioatehydroiodide salt were used to generate final products 11 and 12.

A synthetic route to target compounds 64 and 65, (FIG. 2, in accordancewith structure III) is shown in Scheme 9. Reductive amination3-nitrobenzaldehyde with 2-(3-fluorophenyl)ethanamine generatedintermediate 58. Alternatively, the amino group of 58 was protected withBoc and ethyl groups. Nitro group reduction and coupling with methylthiophene-2-carbimidothioate hydroiodide salt were performed underconditions similar to those adopted for the synthesis of compound 1(e.g., Scheme 1), giving intermediate 63 and target product 64,respectively. Deprotection of the Boc group of 63 generated finalproduct 65.

With reference to FIG. 3 and Scheme 10, below, the synthesis ofcompounds QJ-II-183 and QJ-II-203 followed the same procedure, but forchoice of appropriate starting material. The commercially availablestarting amino compound was protected by dimethylpyrrole followed by aone-step ether synthesis, connecting two heads at the same time. Thenitro compound was reduced to amine by hydrazine in the presence ofRaney-Ni. The amine compound was then reacted with amidothiol etherQJ-II-178, adding the thiophene fragments to each head. After theremoval of protecting group under microwave conditions, the finalproduct was obtained in a 63% yield.

With reference to Scheme 11, the synthesis of compounds QJ-II-187,QJ-II-194, QJ-II-195 and QJ-II-199 followed the same procedure but forchoice of appropriate starting material. The commercially availableBoc-protected prolinyl analogue was reduced to the alcohol by LiBH₄ in aquantitative yield. Then, an ether synthesis linked the two heads via aone-step reaction. The nitro compound was reduced to amine by hydrazinein the presence of Raney-Ni in a 97% yield. The amine compound was thenreacted with amidothiol ether QJ-II-178, adding the thiophene fragmentsto each head. The final product was obtained in a quantitative yieldafter removing the Boc protecting group under acidic conditions.

With reference to Scheme 12, the synthesis of QJ-III-08 (FIG. 3) beginswith a Mitsunobu reaction, coupling the first nitrophenyl group to theprolinyl linker moiety. After reducing the methyl ester to an alcoholwith LiBH₄, the second nitrophenyl group was coupled via a secondMitsunobu reaction. Upon Raney-Ni and hydrazine reduction of the nitrogroups, the resulting compound was ready to react with amidothiol etherQJ-II-178 to provide the double-headed thiophene scaffold. The finalcompound was obtained after removal of the Boc protecting group.

Compound QJ-III-33 (FIG. 3) is very similar to QJ-III-08, but forpara-substitution of the second thiophene head (instead ofmeta-substitution) with respect to the linker moiety. With reference toScheme 13, the same first two steps of the QJ-III-08 synthesis areemployed, but the second Mitsunobu reaction uses para-substitutednitrophenol. The remaining synthetic steps follow those for QJ-III-08with comparable yields.

With reference to Scheme 14, the synthesis of compound QJ-III-07 sharesa key intermediate with that of the two target compounds of Schemes 12and 13; that is, meta-substituted nitrophenyl alcohol. However, insteadof a Mitsunobu reaction, an ether synthesis was used to couple thefluorobenzyl group to the prolinyl linker moiety. The resulting compoundwas reacted with amidothiol ether QJ-II-178 to provide the asymmetricdouble-headed scaffold. With the removal of the Boc protecting group,the final compound was obtained in 85% yield.

Compound QJ-III-21 is similar in structure to QJ-III-07 (see, FIG. 3),but for transposition of the two opposed head groups. With reference toScheme 15, coupling of the fluorobenzyl group to the prolinyl linker viaan ether synthesis is achieved before Mitsunobu reaction with thenitrophenyl group. The remaining synthetic steps are the same asemployed for QJ-III-07, with similar yields.

With respect to either of the compounds, compositions and/or methods ofthe present invention, such compounds or moieties thereof can suitablycomprise, consist of or consist essentially of any of the respectiveaforementioned moieties and/or substituents thereof. Each such compoundor moiety/substituent thereof is compositionally distinguishable,characteristically contrasted and can be practiced in conjunction withthe present invention separate and apart from another. Accordingly, itcan also be understood that the inventive compounds, compositions and/ormethods, as illustratively enclosed disclosed herein, can be practicedor utilized in the absence of any one compound, moiety and/orsubstituent, such compound, moiety, and/or substituent which may or maynot be disclosed, referenced or inferred herein, the absence of whichmay not be specifically disclosed, referenced or inferred herein.

The present invention can also, as would be understood by those skilledin the art, be extended to or include methods using or in conjunctionwith a pharmaceutical composition comprising a compound of the sortdescribed herein and a physiologically or otherwise suitableformulation. In some embodiments, the present invention includes one ormore NOS inhibitors, as set forth above, formulated into a compositiontogether with one or more physiologically tolerable or acceptablediluents, carriers, adjuvants or vehicles that are collectively referredto herein as carriers. Compositions suitable for NOS contact oradministration can comprise physiologically acceptable sterile aqueousor nonaqueous solutions, dispersions, suspensions or emulsions. Theresulting compositions can be, in conjunction with the various methodsdescribed herein, for administration or contact with a human/mammaliannitric oxide synthase expressed or otherwise present therein. Whether ornot in conjunction with a pharmaceutical composition, “contacting” meansthat a nitric oxide synthase and one or more inhibitor compounds of thisinvention are brought together for purpose of binding and/or complexingsuch a compound to the enzyme. Amounts of a compound effective toinhibit a nitric oxide synthase may be determined empirically, andmaking such determinations is within the skill in the art. Inhibition orotherwise affecting or modulating nitric oxide synthase activityincludes both reduction and/or mitigation, as well as elimination of NOSactivity and/or nitric oxide production.

It is understood by those skilled in the art that dosage amount willvary with the activity of a particular inhibitor compound, diseasestate, route of administration, duration of treatment, and like factorswell-known in the medical and pharmaceutical arts. In general, asuitable dose will be an amount which is the lowest dose effective toproduce a therapeutic or prophylactic effect. If desired, an effectivedose of such a compound, pharmaceutically-acceptable salt thereof, orrelated composition may be administered in two or more sub-doses,administered separately over an appropriate period of time.

Methods of preparing pharmaceutical formulations or compositions includethe step of bringing an inhibitor compound into association with acarrier and, optionally, one or more additional adjuvants oringredients. For example, standard pharmaceutical formulation techniquescan be employed, such as those described in Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa.

Regardless of composition or formulation, those skilled in the art willrecognize various avenues for medicament administration, together withcorresponding factors and parameters to be considered in rendering sucha medicament suitable for administration. Accordingly, with respect toone or more non-limiting embodiments, the present invention provides foruse of one or more neuronal nitric oxide synthase inhibitor compoundsfor the manufacture of a medicament for therapeutic use in the treatmentof human disease states implicating NOS activity and/or nitric oxideproduction, including but not limited to the treatment ofneurodegenerative diseases.

In accordance with this invention, various other compounds can beprepared using synthetic techniques of the sort described herein orstraight forward modifications thereof, such modifications as would beunderstood by those skilled in the art made aware of this invention,such compounds, techniques and/or modifications limited only bycommercially- or synthetically-available starting materials andreagents. (See, e.g., FIGS. 2 and 4.)

EXAMPLES OF THE INVENTION

The following non-limiting examples and data illustrate various aspectsand features relating to the compounds and/or methods of the presentinvention, including the characterization and testing of variousselective inhibitor compounds, as are available through the syntheticmethodologies described herein. In comparison with the prior art, thepresent compounds provide results that are surprising, unexpected andcontrary thereto. While the utility of this invention is illustratedthrough the use of several non-limiting compounds, moieties thereof andsubstituents thereto, it will be understood by those skilled in the artthat comparable results are obtainable with various other compounds,moieties and/or substituents, as are commensurate with the scope of thisinvention.

Example 1

With reference to FIG. 1 and schemes 1-9, compounds I-12 and 64-65 werecharacterized as provided below.

Example 1aN,N′-([1,1′-biphenyl]-3,3′-diyl)bis(thiophene-2-carboximidamide) (1)

¹H NMR (500 MHz, DMSO-d6): δ 8.24-8.16 (m, 4H), 7.90-7.80 (m, 4H), 7.70(t, J=8.0 Hz, 2H), 7.51 (d, J=4.5 Hz, 2H), 7.45-7.38 (m, 2H). ¹³C NMR(125 MHz, DMSO-d6): δ 156.61, 140.59, 135.37, 134.96, 134.59, 130.65,129.07, 128.62, 126.63, 125.25, 124.12.

Example 1b(E)-N,N′-(ethane-1,2-diylbis(3,1-phenylene))bis(thiophene-2-carboximidamide)(2)

¹H NMR (500 MHz, MeOD): δ 8.09 (t, J=5.0 Hz, 4H), 7.59-7.27 (m, 10H),6.82 (s, 2H). ¹³C NMR (125 MHz, MeOD): δ 159.37, 140.56, 135.90, 135.61,135.31, 131.57, 131.34, 130.60, 130.02, 127.33, 125.89.

Example 1cN,N′-(ethane-1,2-diylbis(3,1-phenylene))bis(thiophene-2-carboximidamide)(3)

¹H NMR (500 MHz, MeOD): δ 8.17-8.02 (m, 4H), 7.56-7.51 (t, J=7.5 Hz,2H), 7.45-7.38 (m, 6H), 7.36-7.30 (d, J=7.5 Hz, 2H), 3.10 (s, 4H). ¹³CNMR (125 MHz, MeOD): δ 159.57, 145.63, 140.43, 135.40, 135.11, 131.52,130.56, 130.00, 126.82, 124.43, 38.32.

Example 1dN-(3-(1-hydroxy-2-(3-(thiophene-2-carboximidamido)phenoxy)ethyl)phenyl)thiophene-2-carboximidamide(4)

¹H NMR (500 MHz, DMSO-d6): δ 8.28-8.10 (m, 3H), 8.09-7.99 (m, 1H),7.97-7.92 (m, 1H), 7.59-7.52 (m, 2H), 7.46 (t, J=8.5 Hz, 1H), 7.42-7.34(m, 3H), 7.33-7.25 (m, 1H), 7.11-6.97 (m, 2H), 5.03 (m, 1H), 4.20-4.01(m, 1H), 3.80-3.65 (m, 1H). ¹³C NMR (126 MHz, DMSO-d6): δ 159.34,156.89, 156.41, 144.28, 144.20, 140.84, 134.86, 134.62, 134.34, 130.78,129.63, 129.01, 128.53, 128.30, 126.28, 124.35, 123.35, 117.73, 114.78,111.81, 72.97, 70.11.

Example 1eN,N′-((ethane-1,2-diylbis(oxy))bis(3,1-phenylene))bis(thiophene-2-carboximidamide)(5)

¹H NMR (500 MHz, MeOD): δ 8.16-8.06 (m, 4H), 7.54 (t, J=8.0 Hz, 2H),7.40 (t, J=4.5 Hz, 2H), 7.20-7.12 (m, 4H), 7.09 (d, J=8.0 Hz, 2H), 4.46(s, 4H). ¹³C NMR (125 MHz, MeOD): δ 161.61, 159.39, 147.54, 136.67,135.47, 135.16, 132.49, 129.99, 119.00, 116.52, 113.01, 68.19.

Example 1fN,N′-((propane-1,3-diylbis(oxy))bis(3,1-phenylene))bis(thiophene-2-carboximidamide)(6)

¹H NMR (500 MHz, DMSO-d6): δ 8.19 (d, J=6.0 Hz, 1H), 8.14 (d, J=4.0 Hz,1H), 7.47 (t, J=8.0 Hz, 1H), 7.39 (t, J=4.0 Hz, 1H), 7.14-7.08 (s, 1H),7.08-6.98 (m, 1H), 4.20 (t, J=6.0 Hz, 2H), 2.23 (t, J=6.0 Hz, 1H). ¹³CNMR (125 MHz, DMSO-d6): δ 159.33, 156.48, 147.90, 135.61, 134.86,134.53, 130.85, 128.57, 117.72, 114.69, 111.69, 64.41, 28.38.

Example 1gN-(3-(2-((3-(thiophene-2-carboximidamido)benzyl)amino)ethyl)phenyl)thiophene-2-carboximidamide(7)

¹H NMR (500 MHz, MeOD): δ 8.15-8.05 (m, 4H), 7.79 (s, 1H), 7.73-7.69 (m,2H), 7.63-7.54 (m, 2H), 7.53-7.46 (m, 2H), 7.44-7.36 (m, 3H), 4.40 (s,2H), 3.46 (t, J=7.5 Hz, 2H), 3.23 (t, J=7.5 Hz, 2H). ¹³C NMR (125 MHz,MeOD): δ 159.44, 140.62, 136.25, 135.71, 135.51, 135.40, 135.36, 135.17,134.97, 132.33, 132.06, 131.83, 130.79, 130.04, 130.01, 128.59, 127.89,127.42, 125.69, 51.90, 43.75, 33.00.

Example 1hN-(4-(2-((3-(thiophene-2-carboximidamido)benzyl)amino)ethyl)phenyl)thiophene-2-carboximidamide(8)

¹H NMR (500 MHz, MeOD): δ 8.15-8.05 (m, 4H), 7.79 (s, 1H), 7.71 (d,J=5.0 Hz, 2H), 7.58 (d, J=8.0 Hz, 3H), 7.48 (d, J=8.0 Hz, 2H), 7.44-7.37(m, 2H), 4.40 (s, 2H), 3.47-3.40 (t, J=7.5 Hz, 2H), 3.26-3.19 (t, J=7.5Hz, 2H). ¹³C NMR (125 MHz, MeOD): δ 164.83, 142.46, 139.16, 135.72,135.49, 135.42, 135.11, 134.96, 132.35, 132.33, 132.05, 131.82, 130.18,130.05, 130.00, 128.58, 127.92, 127.41, 124.66, 51.87, 43.75, 32.89.

Example 1iN-(3-((pyrrolidin-3-yloxy)methyl)phenyl)thiophene-2-carboximidamide (9)

¹H NMR (500 MHz, MeOD): δ 8.13-8.02 (m, 4H), 7.61 (t, J=7.5 Hz, 2H),7.57-7.48 (m, 4H), 7.47-7.36 (m, 4H), 4.69 (s, 4H), 3.57-3.51 (m, 1H),3.47-3.41 (m, 4H), 3.38-3.34 (m, 2H), 2.39-2.27 (m, 2H), 2.21-2.07 (m,2H). ¹³C NMR (125 MHz, MeOD): δ 157.98, 145.72, 142.06, 135.70, 135.50,135.15, 131.60, 130.01, 129.29, 126.04, 125.79, 78.61, 71.02, 51.79,45.13, 31.40.

Example 1jN-(3-(2-(pyrrolidin-3-ylamino)ethyl)phenyl)thiophene-2-carboximidamide(10)

¹H NMR (500 MHz, MeOD): δ 8.14-8.06 (m, 2H), 7.61 (t, J=8.0 Hz, 1H),7.54-7.48 (m, 2H), 7.44-7.37 (m, 2H), 3.80-3.74 (m, 2H), 3.46-3.40 (m,3H), 3.24-3.19 (m, 2H), 2.68-2.53 (m, 1H), 2.43-2.31 (m, 1H). ¹³C NMR(125 MHz, MeOD): δ 159.51, 140.25, 139.01, 135.52, 135.17, 132.17,130.97, 130.25, 130.01, 127.58, 125.95, 68.15, 57.65, 48.00, 45.89,33.20, 28.70.

Example 1kN-(3-((5-(dimethylamino)naphthalene-1-sulfonamido)methyl)phenyl)thiophene-2-carboximidamide(11)

¹H NMR (500 MHz, DMSO-d6): δ 8.65 (t, J=6.0 Hz, 1H), 8.53 (d, J=8.5 Hz,1H), 8.39 (d, J=8.5 Hz, 1H), 8.20 (d, J=4.5 Hz, 1H), 8.18-8.11 (m, 2H),7.66 (td, J=8.0, 4.5 Hz, 2H), 7.45-7.38 (m, 2H), 7.32-7.24 (m, 3H), 4.11(d, J=6.0 Hz, 2H), 2.88 (s, 6H). ¹³C NMR (125 MHz, DMSO-d6): δ 156.33,152.08, 145.14, 143.72, 140.09, 136.00, 134.90, 134.54, 134.33, 129.73,129.28, 128.91, 128.60, 128.37, 127.90, 127.08, 124.16, 124.10, 123.87,123.83, 45.41, 45.19.

Example 11N-(4-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)phenyl)thiophene-2-carboximidamide(12)

¹H NMR (500 MHz, DMSO-d6): δ 8.55 (d, J=8.5 Hz, 1H), 8.37 (d, J=8.5 Hz,1H), 8.18 (d, J=5.0 Hz, 1H), 8.16-8.11 (m, 3H), 7.73-7.58 (m, 2H),7.46-7.35 (m, 2H), 7.34-7.25 (m, 3H), 3.11-2.99 (m, 2H), 2.91 (s, 6H),2.75-2.70 (m, 2H). ¹³C NMR (125 MHz, DMSO-d6): δ 156.45, 149.27, 141.62,138.91, 135.92, 134.79, 134.42, 132.64, 130.09, 129.16, 129.06, 128.97,128.57, 128.36, 127.82, 125.44, 45.24, 34.88.

Example 1mN-(3-((ethyl(3-fluorophenethyl)amino)methyl)phenyl)thiophene-2-carboximidamide(64)

¹H NMR (400 MHz, CD₃OD): δ 7.85-7.72 (m, 2H), 7.43 (t, J=7.5 Hz, 1H),7.31-7.17 (m, 4H), 7.12 (dt, J=7.5, 1.0 Hz, 1H), 7.02 (dt, J=7.5, 1.0Hz, 1H), 6.97 (dt, J=9.0, 2.5 Hz, 1H), 6.88 (td, J=9.0, 2.5 Hz, 1H),3.96 (s, 2H), 2.99-2.82 (m, 6H), 1.18 (t, J=7.0 Hz, 3H). ¹³C NMR (100MHz, CD₃OD): δ 164.05, 161.62, 141.73 (d, J=7.5 Hz), 135.05, 130.92,130.00, 129.91, 129.82, 129.80, 127.70, 126.86, 124.80, 124.44, 124.41,123.27, 115.19 (d, J=20 Hz), 112.72 (d, J=20 Hz), 109.99, 56.82, 53.51,47.10, 31.10, 9.67.

Example 1nN-(3-(((3-fluorophenethyl)amino)methyl)phenyl)thiophene-2-carboximidamide(65)

¹H NMR (400 MHz, CD₃OD): δ 8.18-8.04 (m, 2H), 7.81-7.69 (m, 2H), 7.65(t, J=7.5 Hz, 1H), 7.58-7.50 (m, 1H), 7.41-7.28 (m, 2H), 7.18-7.06 (m,2H), 7.03-6.93 (m, 1H), 4.37 (s, 2H), 3.42-3.31 (m, 2H), 3.20-3.10 (m,2H). ¹³C NMR (100 MHz, CD₃OD): δ 164.17, 161.74 (s), 139.31 (d, J=7.5Hz), 134.72, 134.45, 134.22, 133.50, 130.87, 130.55, 130.34 (d, J=7.5Hz), 128.66, 127.22, 126.47, 124.49, 124.46, 115.30 (d, J=20 Hz), 113.56(d, J=20 Hz), 109.99, 50.38, 48.27, 31.49 (d, J=2.0 Hz).

The compounds of FIG. 3, (Schemes 10-15) were characterized, as providedbelow in Examples 2-11.

Example 2

(R)-QJ-II-183

¹H NMR (500 MHz, CDCl₃) δ 7.35-7.32 (m, 4H), 7.25-7.19 (m, 2H),6.99-6.94 (m, 4H), 6.90 (s, 2H), 6.83 (d, J=7.5 Hz, 2H), 4.88 (br, 4H),4.60 (d, J=5.0 Hz, 1H), 4.50 (d, J=5.0 Hz, 1H), 4.44 (s, 2H), 3.52-3.48(m, 3H), 2.80-2.70 (m, 2H); ¹³C NMR (126 MHz, DMSO) δ ¹³C NMR (126 MHz,CDCl₃) δ 149.88, 149.09, 140.51, 139.90, 139.51, 129.55, 128.96, 127.30,126.05, 122.76, 122.64, 121.33, 121.30, 121.15, 79.28, 73.36, 72.03,70.54, 50.73, 43.26 ppm; MS (ESI): 520.8 (M+H)⁺.

Example 3

(S)-QJ-II-203

¹H NMR (500 MHz, CDCl₃) δ 7.36-7.32 (m, 4H), 7.25-7.19 (m, 2H),6.99-6.94 (m, 4H), 6.90 (s, 2H), 6.82 (d, J=7.5 Hz, 2H), 4.88 (br, 4H),4.60 (d, J=5.0 Hz, 1H), 4.50 (d, J=5.0 Hz, 1H), 4.44 (s, 2H), 3.52-3.48(m, 3H), 2.80-2.70 (m, 2H); ¹³C NMR (126 MHz, DMSO) δ ¹³C NMR (126 MHz,CDCl₃) δ 149.88, 149.10, 140.51, 139.90, 139.51, 129.55, 128.96, 127.30,126.05, 122.76, 122.64, 121.33, 121.30, 121.16, 79.28, 73.36, 72.03,70.54, 50.72, 43.26 ppm; MS (ESI): 520.6 (M+H)⁺.

Example 4

(S,S)-QJ-II-195

¹H NMR (500 MHz, CDCl₃) δ 7.42 (t, J=5.0 Hz, 2H), 7.40 (d, J=4.0 Hz,1H), 7.36 (d, J=4.0 Hz, 1H), 7.30 (t, J=7.5 Hz, 2H), 7.08-7.00 (m, 4H),6.95 (d, J=7.5 Hz, 2H), 6.91 (d, J=7.5 Hz, 2H), 4.90 (br, 2H), 4.53 (s,2H), 4.44 (d, J=5.0 Hz, 2H), 4.13-4.11 (m, 1H), 3.56-3.53 (m, 2H),3.30-3.27 (m, 1H), 3.10 (d, J=5.0 Hz, 1H), 2.88 (dd, J=5.0 Hz, J=10.0Hz, 1H), 2.13-2.09 (m, 1H), 1.81 (br, 2H), 1.65-1.60 (m, 1H); δ ¹³C NMR(126 MHz, CDCl₃) δ 149.83, 149.75, 149.02, 140.56, 140.50, 139.82,139.69, 129.53, 128.98, 128.93, 127.28, 125.97, 122.70, 122.54, 121.21,121.09, 79.90, 73.18, 72.88, 70.90, 57.86, 52.58, 50.85, 35.19 ppm; MS(ESI): 546.3 (M+H)⁺.

Example 5

(R,R)-QJ-II-187

¹H NMR (500 MHz, CDCl₃) δ 7.42 (t, J=5.0 Hz, 2H), 7.40 (d, J=4.0 Hz,1H), 7.36 (d, J=4.0 Hz, 1H), 7.30 (t, J=7.5 Hz, 2H), 7.08-7.00 (m, 4H),6.95 (d, J=7.5 Hz, 2H), 6.91 (d, J=7.5 Hz, 2H), 4.90 (br, 2H), 4.53 (s,2H), 4.44 (d, J=5.0 Hz, 2H), 4.13-4.11 (m, 1H), 3.56-3.53 (m, 2H),3.30-3.27 (m, 1H), 3.10 (d, J=5.0 Hz, 1H), 2.88 (dd, J=5.0 Hz, J=10.0Hz, 1H), 2.13-2.09 (m, 1H), 1.81 (br, 2H), 1.65-1.60 (m, 1H); δ ¹³C NMR(126 MHz, CDCl₃) δ 149.83, 149.75, 149.02, 140.56, 140.50, 139.82,139.69, 129.53, 128.98, 128.93, 127.28, 125.97, 122.70, 122.54, 121.21,121.09, 79.90, 73.18, 72.88, 70.90, 57.86, 52.58, 50.85, 35.19 ppm; MS(ESI): 546.2 (M+H)⁺.

Example 6

(S,R)-QJ-II-194

¹H NMR (500 MHz, CDCl₃) δ 7.43 (d, J=5.0 Hz, 2H), 7.40 (d, J=4.5 Hz,2H), 7.32 (t, J=7.5 Hz, 2H), 7.07 (t, J=4.5 Hz, 2H), 7.03 (d, J=7.5 Hz,2H), 6.97 (s, 2H), 6.91 (d, J=7.5 Hz, 2H), 4.87 (br, 2H), 4.51 (d, J=5.0Hz, 2H), 4.47 (d, J=5.0 Hz, 2H), 4.13-4.11 (m, 1H), 3.55-3.53 (m, 1H),3.47-3.45 (m, 1H), 3.42-3.38 (m, 1H), 3.09-2.95 (m, 1H), 2.00 (dd, J=7.5Hz, J=13.5 Hz, 1H), 1.84 (br, 2H), 1.65-1.59 (m, 1H); δ ¹³C NMR (126MHz, CDCl₃) δ 149.75, 148.97, 140.51, 139.85, 139.75, 129.56, 129.51,128.99, 127.29, 125.97, 122.65, 122.54, 121.14, 121.11, 121.04, 79.95,73.39, 73.20, 70.85, 56.73, 52.47, 50.85, 35.15; MS (ESI): 546.2 (M+H)⁺.

Example 7

(R,S)-QJ-II-199

¹H NMR (500 MHz, CDCl₃) δ 7.43 (d, J=5.0 Hz, 2H), 7.40 (d, J=4.5 Hz,2H), 7.32 (t, J=7.5 Hz, 2H), 7.07 (t, J=4.5 Hz, 2H), 7.02 (d, J=7.5 Hz,2H), 6.97 (s, 2H), 6.91 (d, J=7.5 Hz, 2H), 4.87 (br, 2H), 4.51 (d, J=5.0Hz, 2H), 4.47 (d, J=5.0 Hz, 2H), 4.13-4.10 (m, 1H), 3.55-3.53 (m, 1H),3.47-3.45 (m, 1H), 3.42-3.38 (m, 1H), 3.09-2.95 (m, 1H), 2.00 (dd, J=7.5Hz, J=13.5 Hz, 1H), 1.84 (br, 2H), 1.65-1.59 (m, 1H); δ ¹³C NMR (126MHz, CDCl₃) δ 149.75, 148.97, 140.51, 139.86, 139.75, 129.56, 129.52,128.99, 127.29, 125.97, 122.65, 122.54, 121.14, 121.11, 121.03, 79.95,73.39, 73.20, 70.86, 56.73, 52.47, 50.85, 35.15; MS (ESI): 546.3 (M+H)⁺.

Example 8

QJ-III-08

¹H NMR (500 MHz, CD₃OD) δ 7.64 (s, 1H), 7.56 (d, J=10.0 Hz, 1H), 7.29(t, J=8.0 Hz, 2H), 7.13 (t, J=12.0 Hz, 1H), 7.00 (t, J=8.0 Hz, 3H), 6.70(dd, J=17.5 Hz, J=8.0 Hz, 1H), 6.61-6.55 (m, 3H), 6.35-6.30 (m, 2H),4.03-3.92 (m, 2H), 3.77-3.70 (m, 1H), 3.37 (s, 2H), 3.15-3.08 (m, 1H),2.25-2.19 (m, 1H), 1.99-1.92 (m, 1H); ¹³C NMR (126 MHz, CD₃OD)δ161.44+161.32, 160.04, 159.87, 153.92, 151.38, 150.14+150.06, 140.96,131.43+131.37, 130.92+130.87, 129.94+129.91, 128.53, 128.41,116.20+116.10, 111.91, 110.92+110.83, 109.86, 109.71+109.65, 106.44,105.43, 104.02, 103.01, 79.18+78.92, 71.28+71.04, 57.87+57.82, 53.26,36.68+36.66 ppm. MS (ESI): 518.3 (M+H)⁺.

Example 9

III-33

¹H NMR (500 MHz, CD₃OD) δ 7.53-7.50 (m, 2H), 7.46-7.44 (m, 2H),7.03-7.00 (m, 2H), 6.91-6.87 (m, 3H), 6.82-6.80 (m, 2H), 6.59-6.48 (m,1H), 6.45 (s, 1H), 6.24-6.21 (m, 1H), 4.26-4.12 (m, 1H), 3.93-3.85 (m,2H), 3.66-3.62 (s, 1H), 3.01 (dd, J=18.5 Hz, J=12.5 Hz, 1H), 2.31-2.26(m, 1H), 2.15-2.07 (m, 1H), 1.90-1.81 (m, 1H); ¹³C NMR (126 MHz, CD₃OD)δ 164.04, 162.56, 162.38, 159.27, 133.99, 133.47, 132.49, 132.32,130.96, 130.92, 127.13, 119.19, 118.65, 114.46, 113.36, 112.24, 109.02,106.60, 81.74+81.46, 74.17, 60.39, 55.87+55.80, 39.18 ppm. MS (ESI):518.2 (M+H)⁺.

Example 10

QJ-III-07

¹H NMR (500 MHz, CDCl₃) δ 7.45 (dd, J=9.0 Hz, J=5.0 Hz, 2H), 7.25-7.22(m, 1H), 7.15 (d, J=8.0 Hz, 1H), 7.03-6.97 (m, 3H), 6.89 (t, J=8.0 Hz,1H), 6.50 (dd, J=4.0 Hz, J=2.0 Hz, 2H), 6.43 (s, 1H), 4.80 (br, 2H),4.76 (t, J=6.0 Hz, 1H), 4.49-4.43 (m, 2H), 3.57-3.54 (m, 1H), 3.44-3.35(m, 2H), 3.18 (dd, J=12.0 Hz, J=4.0 Hz, 1H), 3.07 (d, J=12.0 Hz, 1H),2.03 (dd, J=14.0 Hz, J=7.0 Hz, 1H), 1.76-1.70 (m, 1H); ¹³C NMR (126 MHz,CDCl₃) δ 163.90, 161.95, 158.67, 150.27, 149.65, 141.01+140.96, 140.45,130.32, 129.93+129.87, 129.04, 127.32, 125.99, 122.94,114.52+114.41+114.35+114.24, 114.08, 110.87, 108.69, 78.18, 73.42,72.52, 56.94, 52.85, 35.66 ppm; MS (ESI): 426.3 (M+H)⁺.

Example 11

QJ-III-21

¹H NMR (500 MHz, CDCl₃) δ 7.37 (d, J=5.0 Hz, 1H), 7.33 (d, J=3.5 Hz,1H), 7.26 (q, J=9.5 Hz, J=7.5 Hz, 1H), 7.16 (t, J=8.0 Hz, 1H), 7.03-6.98(m, 3H), 6.90 (t, J=8.0 Hz, 1H), 6.56-6.48 (m, 2H), 6.25-6.17 (m, 1H),4.77 (br, 2H), 4.43-4.39 (m, 2H), 4.09 (s, 1H), 3.87-3.79 (m, 2H),3.70-3.56 (m, 1H), 3.07-3.04 (m, 2H), 2.05 (dd, J=13.5 Hz, J=7.0 Hz,1H), 1.69-1.64 (m, 1H); ¹³C NMR (126 MHz, CDCl₃) δ 163.93, 161.97,160.06, 150.21+149.60, 147.71, 141.09+141.03, 140.49, 130.23,129.96+129.90+129.02, 127.30, 125.92, 122.86,114.53+114.38+114.36+114.20, 109.89+108.04, 107.85, 104.59, 101.70,80.18, 71.01, 70.11, 56.20, 52.51, 35.13 ppm; MS (ESI): 426.6 (M+H)⁺.

Example 12

NOS inhibition assays were undertaken for compounds I-12 and 64-65 ofFIG. 1 and for the compounds of FIG. 3, and the results are provided inTables 1 and 2, respectively.

TABLE 1 Inhibition of NOS isozymes by synthetic inhibitors.^(a) Ki (μM)Selectivity^(b) No. nNOS iNOS eNOS n/i n/e 1 0.787 66.4 177.3 84 225 26.470 19.4 491.7 3 76 3 0.739 103.8 66.0 141 89 4 0.819 10.1 5.2 12 6 50.130 66.8 13.7 513 105 6 0.237 12.6 4.0 53 17 7 0.005 1.3 2.2 235 407 80.005 1.7 2.7 312 495 9 0.139 36.5 15.8 263 113 10 0.131 6.9 40.8 53 31111 0.498 34.3 115.4 69 232 12 0.243 142.4 7.28 586 30 64 0.073 12.1 21.7166 297 65 0.011 1.6 0.9 145 82 ^(a)The compounds were evaluated for invitro inhibition against three NOS isozymes: rat nNOS, bovine eNOS andmarine iNOS using known literature methods. (Hevel, J. M.; Marietta, M.A. Nitric-oxide synthase assays. Method Enzymol. 1994, 233, 250-258).^(b)Each of n/e and n/i is the inverse of the ratio of K_(i) (eNOS oriNOS) to K_(i) (nNOS).

TABLE 2 Inhibition of NOS isozymes by synthetic inhibitors.^(a) Ki [μM]Selectivity^(b) Compounds nNOS eNOS iNOS n/e n/i QJ-II-183 0.0590 12.448.11 210 137 QJ-II-203 0.0147 16.68 4.73 1134 322 QJ-II-187 0.0281 3.631.99 130 71 QJ-II-194 0.0132 2.47 1.11 190 85 QJ-II-195 0.0221 1.45 2.11658 96 QJ-II-199 0.0214 3.17 1.23 151 58 QJ-III-07 0.0684 5.89 6.17 8690 QJ-III-08 0.0282 3.34 2.42 119 86 QJ-III-21 0.101 1.86 9.28 18 92QJ-III-33 0.0270 1.15 4.40 142 163 ^(a)K_(m) values of rat nNOS, 1.3 μM;murine iNOS, 8.2 μM; bovine eNOS, 1.7 μM). Ki = IC₅₀/ (1 + [S]/K_(M)).^(b)Each of n/e and n/i is the inverse of the ratio of K_(i) (eNOS oriNOS) to K_(i) (nNOS).

We claim:
 1. A compound of a formula

wherein L is a divalent CH₂CH₂ moiety with a meta-relationship to eachsaid thiophene-2-carboximidamide moiety of said compound; or a saltthereof.